Exploring the underlying mechanisms of fisetin in the treatment of hepatic insulin resistance via network pharmacology and in vitro validation

Abstract

Objective: To characterize potential mechanisms of fisetin on hepatic insulin resistance (IR) using network pharmacology and in vitro validation.

Methods: Putative targets of fisetin were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, whereas the potential genes of hepatic IR were obtained from GeneCards database. A protein-protein interaction (PPI) network was constructed according to the intersection targets of fisetin and hepatic IR using the Venn diagram. The biological functions and potential pathways related to genes were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Cell experiments were also conducted to further verify the mechanism of fisetin on hepatic IR.

Results: A total of 118 potential targets from fisetin were associated with hepatic IR. The areas of nodes and corresponding degree values of TP53, AKT1, TNF, IL6, CASP3, CTNNB1, JUN, SRC, epidermal growth factor receptor (EGFR), and HSP90AA1 were larger and could be easily found in the PPI network. Furthermore, GO analysis revealed that these key targets were significantly involved in multiple biological processes that participated in oxidative stress and serine/threonine kinase activity. KEGG enrichment analysis showed that the PI3K/AKT signaling pathway was a significant pathway involved in hepatic IR. Our in vitro results demonstrated that fisetin treatment increased the expressions of EGFR and IRS in HepG2 and L02 cells under normal or IR conditions. Western blot results revealed that p-AKT/AKT levels were significantly up-regulated, suggesting that fisetin was involved in the PI3K/AKT signaling pathway to regulate insulin signaling.

Conclusion: We explored the pharmacological actions and the potential molecular mechanism of fisetin in treating hepatic IR from a holistic perspective. Our study lays a theoretical foundation for the development of fisetin for type 2 diabetes.

Keywords: Epidermal growth factor receptor; Fisetin; Hepatic insulin resistance; Network pharmacology; PI3K/AKT signaling.

Fig. 1

The design profile of the current study. Hepatic IR: hepatic insulin resistance; BP: biological process; CC: cellular component; MF: molecular function. The green box indicates Schematic structure of EGFR. On the left side of Fisetin is its chemical structure

Fig. 2

A Venny diagram of the fisetin targets and hepatic IR-related genes. B The PPI network of 118 intersection targets. C The top 20 hub gene networks of fisetin for hepatic IR treatment

Fig. 3

GO and KEGG analysis of the 118 intersection targets from fisetin were associated with hepatic IR. A GO biological process terms. B GO cellular component terms. C GO molecular function terms. D KEGG pathway. The size of each dot corresponds to the number of gene annotated in the entry, and the color of each dot corresponds to the corrected p-value

Fig. 4

GO and KEGG analysis of the top 20 hub genes for fisetin in the treatment of hepatic IR. A GO biological process terms. B GO cellular component terms. C GO molecular function terms. D KEGG pathway. The size of each dot corresponds to the number of gene annotated in the entry, and the color of each dot corresponds to the corrected p-value

Fig. 5

A The compound-targets-disease network. The yellow arrow represented fisetin, the red diamond represented hepatic IR, and blue circle represented the targets. B PI3K/AKT signaling pathway. The red lines show the possible signaling pathways through which fisetin acted on the hepatic IR

Fig. 6

Effects of fisetin on EGFR and key factors (insulin receptor substrate (IRS) and AKT) in insulin signaling pathway in L02 and HepG2 cells. A Relative expression of RNA and proteins related to fisetin treatment in L02 normal or IR cells. B Relative expression of RNA and proteins related to fisetin treatment in HepG2 normal or IR cells. C Relative expression of RNA and proteins related to EGFR activator (NSC 228155) in L02 IR cells. Control represents untreated L02 cells. * P < 0.05, ** P < 0.01 and *** P < 0.001